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Study of a new derivatizing reagent that improves the analysis of amino acids by HPLC with fluorescence detection: application to hydrolyzed rape bee pollen

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Abstract

A simple and sensitive method for evaluating the chemical compositions of protein amino acids, including cystine (Cys)2 and tryptophane (Try) has been developed, based on the use of a sensitive labeling reagent 2-(11H-benzo[α]-carbazol-11-yl) ethyl chloroformate (BCEC–Cl) along with fluorescence detection. The chromophore of the 1,2-benzo-3,4-dihydrocarbazole-ethyl chloroformate (BCEOC-Cl) molecule was replaced with the 2-(11H-benzo[α]-carbazol-11-yl) ethyl functional group, yielding the sensitive fluorescence molecule BCEC–Cl. The new reagent BCEC–Cl could then be substituted for labeling reagents commonly used in amino acid derivatization. The BCEC–amino acid derivatives exhibited very high detection sensitivities, particularly in the cases of (Cys)2 and Try, which cannot be determined using traditional labeling reagents such as 9-fluorenyl methylchloroformate (FMOC-Cl) and ortho-phthaldialdehyde (OPA). The fluorescence detection intensities for the BCEC derivatives were compared to those obtained when using FMOC-Cl and BCEOC-Cl as labeling reagents. The ratios I BCEC/I BCEOC = 1.17–3.57, I BCEC/I FMOC = 1.13–8.21, and UVBCEC/UVBCEOC = 1.67–4.90 (where I is the fluorescence intensity and UV is the ultraviolet absorbance). Derivative separation was optimized on a Hypersil BDS C18 column. The detection limits calculated from 1.0 pmol injections, at a signal-to-noise ratio of 3, ranged from 7.2 fmol for Try to 8.4 fmol for (Cys)2. Excellent linear responses were observed, with coefficients of >0.9994. When coupled with high-performance liquid chromatography, the method established here allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids including (Cys)2 and Try from bee-collected pollen (bee pollen) samples.

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References

  1. Silva TMS, Camara CA, Lins ACS, Barbosa-Filho JM, Silva EMS, Freitas BM, Santos FAR (2006) J Food Compos Anal 19:507–551

    Article  CAS  Google Scholar 

  2. Dobson HM, Peng Y-S (1997) J Insect Physiol 40:89–100

    Article  Google Scholar 

  3. Fiamegos YC, Stalikas CD (2006) J Chromatogr A 1110:66–72

    Article  CAS  Google Scholar 

  4. Nozal MJ, Bernal JL, Toribio ML, Diego JC, Ruiz A (2004) J Chromatogr A 1047:137–146

    Article  CAS  Google Scholar 

  5. Hope JL, Prazen BJ, Nilsson EJ, Lidstrom ME, Synovec RE (2005) Talanta 65:380–388

    Article  CAS  Google Scholar 

  6. López-Cervantes J, Sánchez-Machado DI, Rosas-Rodríguez JA (2006) J Chromatogr A 1105:106–110

    Article  Google Scholar 

  7. Song Y, Shenwu M, Zhao S, Hou DY, Liu Y-M (2005) J Chromatogr A 1091:102–109

    Article  CAS  Google Scholar 

  8. Kang X, Xiao J, Huang X, Gu ZZ (2006) Clin Chim Acta 366:352–356

    Article  CAS  Google Scholar 

  9. Kubáň P, Hauser PC (2006) J Chromatogr A 1128:97–104

    Google Scholar 

  10. Inoue H, Iguchi H, Kono A, Tsuruta Y (1999) J Chromatogr A 724:221–230

    CAS  Google Scholar 

  11. Kirschbaum J, Luckas B, Beinert W-D (1994) J Chromatogr A 661:193–199

    Article  CAS  Google Scholar 

  12. Hirano H, Wittmann-Liebold B (1986) Biol Chem Hoppe-Seyler 367:1259–1265

    CAS  Google Scholar 

  13. Ishida J, Yamaguchi M, Iwata T, Nakamura M (1989) Anal Chim Acta 223:319–324

    Article  CAS  Google Scholar 

  14. Liu H, Cho B-Y, Krull IS, Cohen SA (2001) J Chromatogr A 927:77–89

    Article  CAS  Google Scholar 

  15. Turiák G, Volicer L (1994) J Chromatogr A 668:323–329

    Article  Google Scholar 

  16. Hill WD, Walters FH, Wilson TD, Stuart JD (1979) Anal Chem 51:1338–1341

    Article  CAS  Google Scholar 

  17. Chem RF, Sctt C, Trepman E (1979) Biochim Biophys Acta 576:440–455

    Google Scholar 

  18. Einarrson S, Joseffson B, Lagerkvist S (1983) J Chromatogr 282:609–618

    Article  Google Scholar 

  19. Ahnoff M, Grundevik I, Arfwidsson A, Fonselius J, Bengt AP (1981) Anal Chem 53:485–489

    Article  CAS  Google Scholar 

  20. Einarsson S, Josefsson B, Lagerkvist S (1983) J Chromatogr 282:609–673

    Article  CAS  Google Scholar 

  21. Buratti M, Valla C, Xaiz D, Brambilla G, Colombi A (2002) J Chromatogr B 776:237–243

    Article  CAS  Google Scholar 

  22. Einarsson S, Josefsson B, Moller P, Sanchez D (1987) Anal Chem 59:1191–1195

    Article  CAS  Google Scholar 

  23. Bjöklund J, Einarsson S, Engström A, Grzegorczyk A, Becker H-D (1998) J Chromatogr A 798:1–8

    Article  Google Scholar 

  24. Kovács A, Sarkadi LS, Ganzler K (1999) J Chromatogr A 836:305–313

    Article  Google Scholar 

  25. Liu HJ (1994) J Chromatogr A 670:59–66

    Article  CAS  Google Scholar 

  26. You J, Zhang W, Zhang Q, Zhang L, Yan C, Zhang Y (2002) Anal Chem 74:261–269

    Article  CAS  Google Scholar 

  27. You JM, Chen XM, Zhao X, Suo Y, Wang HL, Li Y, Sun J (2006) Chromatographia 63:337–343

    Article  CAS  Google Scholar 

  28. You JM, Zhao XE, Suo YR, Wang HL, Li YL, Sun J (2006) Anal Chim Acta 565:168–177

    Article  CAS  Google Scholar 

  29. You JM, Ming YF, Shi YW, Zhao XE, Suo YR, Wang HL, Li YL, Sun J (2005) Talanta 68:448–458

    Article  CAS  Google Scholar 

  30. Cohen SA, Michaud DP (1993) Anal Biochem 211:279–287

    Article  CAS  Google Scholar 

  31. Duan H (2000) Organic synthesis manual. Chemical Industry Press, Beijing, China, pp 445–446

  32. Oliveira MM, Salvador MA, Coelho PJ, Carvalho LM (2005) Tetrahedron 61:1681–1691

    Article  CAS  Google Scholar 

  33. You J, Shan Y, Shan Y, Zhen L, Zhang L, Zhang Y (2003) Anal Biochem 313:17–27

    Article  CAS  Google Scholar 

  34. Strydom DJ, Tarr GE, Pan Y-CE, Paxton RJ (1992) In: Angeletti RH (ed) Techniques in protein chemistry III. Academic, San Diego, CA, pp 261–271

  35. Paramás AMG, Bárez JAG, Marcos CC, G-Villanova RJ, Sánchez JS (2006) Food Chem 95:148–156

    Article  Google Scholar 

Download references

Acknowledgment

This work was supported by the National Science Foundation under Grants 20075016 and 30370218.

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Authors and Affiliations

  1. Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining, 810001, China

    Jinmao You, Xianen Zhao & Yourui Suo

  2. Department of Chemistry, Qufu Normal University, Qufu Shandong, 273165, China

    Jinmao You, Lingjun Liu, Wenchen Zhao, Honglun Wang & Yulin Li

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  1. Jinmao You
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  2. Lingjun Liu
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  3. Wenchen Zhao
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  4. Xianen Zhao
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  5. Yourui Suo
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  6. Honglun Wang
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  7. Yulin Li
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Correspondence to Jinmao You.

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You, J., Liu, L., Zhao, W. et al. Study of a new derivatizing reagent that improves the analysis of amino acids by HPLC with fluorescence detection: application to hydrolyzed rape bee pollen. Anal Bioanal Chem 387, 2705–2718 (2007). https://doi.org/10.1007/s00216-007-1155-9

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  • DOI: https://doi.org/10.1007/s00216-007-1155-9

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